Proper regulation of lipid metabolism is central to human health. Disruption of lipid metabolic pathways can lead to a variety of diseases including diabetes and obesity, which represent a substantial public health burden, especially in the last few decades. microRNAs (miRNAs) are small, non-coding RNAs that have emerged as potent and abundant regulators of cholesterol and energy metabolism. Furthermore, dysfunction in miRNA pathways has been implicated in the etiology of a variety of metabolic disorders. Recently, we demonstrated that hepatic miR-29 is aberrantly elevated in animal models of dyslipidemia and diabetes and also showed that miR-29 regulates lipid metabolic pathways in vitro. Subsequent studies in vivo revealed that miR-29 inhibition leads to the suppression of cholesterol and fatty acid synthesis pathways and a dramatic reduction in plasma cholesterol and triglycerides. We confirmed in human hepatoma cells in vitro that inhibition of miR-29 suppresses de novo cholesterol and fatty acid synthesis. Upon further analysis, we showed that miR-29 inhibition in vivo up-regulates hepatic protein levels of the transcription factors (TFs) Ah and Foxo3, both of which are involved in the suppression of lipid synthesis. We also demonstrated that Ahr is likely a direct target of miR-29. Among the known Ahr target genes that were significantly upregulated by miR- 29 inhibition is Fgf21, an important endocrine regulator of lipid homeostasis. We also observed that miR-29 inhibition leads to >10-fold down-regulation of miR-200a, which has been linked previously to metabolic control. Ahr was reported recently as a negative regulator of miR-200a, and our bioinformatic analysis identified a candidate Ahr binding site in the miR-200a promoter. Based on these and related preliminary data, we hypothesize that miR-29 control of lipid homeostasis is mediated in large part by a regulatory cascade driven by Ahr, Foxo3, and miR-200a. In this proposal, we will test this hypothesis with a blend of genomic and molecular strategies, using miR-29 loss- and gain-of-function animal models. Successful completion of these aims will uncover the molecular and biological functions of miR-29 in lipid homeostasis. These findings will lay a strong foundation for future studies focused on the design of miR-29-based research tools and novel therapeutic strategies for metabolic disease.